Ubiquitin binding modulates IAP antagonist-stimulated proteasomal degradation of c-IAP1 and c-IAP2(1).
A family of anti-apoptotic regulators known as IAP (inhibitor of apoptosis) proteins interact with multiple cellular partners and inhibit apoptosis induced by a variety of stimuli. c-IAP (cellular IAP) 1 and 2 are recruited to TNFR1 (tumour necrosis factor receptor 1)-associated signalling complexes, where they mediate receptor-induced NF-kappaB (nuclear factor kappaB) activation. Additionally, through their E3 ubiquitin ligase activities, c-IAP1 and c-IAP2 promote proteasomal degradation of NIK (NF-kappaB-inducing kinase) and regulate the non-canonical NF-kappaB pathway. In the present paper, we describe a novel ubiquitin-binding domain of IAPs. The UBA (ubiquitin-associated) domain of IAPs is located between the BIR (baculovirus IAP repeat) domains and the CARD (caspase activation and recruitment domain) or the RING (really interesting new gene) domain of c-IAP1 and c-IAP2 or XIAP (X-linked IAP) respectively. The c-IAP1 UBA domain binds mono-ubiquitin and Lys(48)- and Lys(63)-linked polyubiquitin chains with low-micromolar affinities as determined by surface plasmon resonance or isothermal titration calorimetry. NMR analysis of the c-IAP1 UBA domain-ubiquitin interaction reveals that this UBA domain binds the classical hydrophobic patch surrounding Ile(44) of ubiquitin. Mutations of critical amino acid residues in the highly conserved MGF (Met-Gly-Phe) binding loop of the UBA domain completely abrogate ubiquitin binding. These mutations in the UBA domain do not overtly affect the ubiquitin ligase activity of c-IAP1 or the participation of c-IAP1 and c-IAP2 in the TNFR1 signalling complex. Treatment of cells with IAP antagonists leads to proteasomal degradation of c-IAP1 and c-IAP2. Deletion or mutation of the UBA domain decreases this degradation, probably by diminishing the interaction of the c-IAPs with the proteasome. These results suggest that ubiquitin binding may be an important mechanism for rapid turnover of auto-ubiquitinated c-IAP1 and c-IAP2.
Blankenship
John W
JW
Department of Protein Engineering, Genentech, Inc., 1 DNA Way, M/S 40, South San Francisco, CA 94080, USA.
Varfolomeev
Eugene
E
Goncharov
Tatiana
T
Fedorova
Anna V
AV
Kirkpatrick
Donald S
DS
Izrael-Tomasevic
Anita
A
Phu
Lilian
L
Arnott
David
D
Aghajan
Mariam
M
Zobel
Kerry
K
Bazan
J Fernando
JF
Fairbrother
Wayne J
WJ
Deshayes
Kurt
K
Vucic
Domagoj
D
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002151:Calorimetry; D002460:Cell Line; D045744:Cell Line, Tumor; D002942:Circular Dichroism; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism; D025821:Polyubiquitin / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance; D025801:Ubiquitin / Q000378:metabolism*; D054875:Ubiquitination; D000097782:NF-kappaB-Inducing Kinase
D051034:Inhibitor of Apoptosis Proteins; D016328:NF-kappa B; D025821:Polyubiquitin; D046988:Proteasome Endopeptidase Complex; D017346:Protein Serine-Threonine Kinases; D047888:Receptors, Tumor Necrosis Factor, Type I; D025801:Ubiquitin; D000097782:NF-kappaB-Inducing Kinase